Use of volumetric absorptive microsampling and parallel reaction monitoring mass spectrometry for tacrolimus blood trough measurements at home in pediatric heart transplant patients.

Background: Measurement of trough levels for calcineurin inhibitors by venipuncture sampling is a mainstay of patient management in solid organ transplant recipients but challenging in pediatric patients. Volumetric Absorptive Microsampling (VAMS) is a patient-friendly, minimally invasive sampling technique to accurately collect blood. An assay for measurement of tacrolimus in blood using VAMS, coupled with parallel reaction monitoring (PRM) mass spectrometry, was validated in pediatric heart transplant patients. Methods: Tacrolimus was measured by a newly developed high-resolution PRM assay and compared with low-resolution tandem mass spectrometry (MRM). Dried blood samples were collected from pediatric heart transplant patients (n = 35) using VAMS devices and a satisfaction survey was completed by patients/guardians. Tacrolimus concentrations were compared across whole liquid blood, dried blood spots, and capillary blood, and shipping stability determined. Results: The PRM assay was linear over a range 1-50 ng/mL, similar to MRM but had greater specificity due to reduced background noise. No significant differences in tacrolimus concentrations were observed between VAMS and venous blood. Tacrolimus dried on VAM tips was stable for 14 days and concentrations were unaffected by postal shipping. The variability in two simultaneously collected at-home patient samples was minimal - average concentration difference was 0.12 ± 0.94 ng/mL (p = 0.6) between paired samples. Conclusion: A high resolution PRM mass spectrometry assay was developed for home-based dried blood collections for therapeutic monitoring of tacrolimus. The advantage of PRM was enhanced specificity and the VAMS devices provided a simple and convenient approach to blood sampling at home in pediatric heart transplant patients.

Pre-operative neutrophil-lymphocyte ratio predicts low cardiac output in children after cardiac surgery.

Neutrophil-lymphocyte ratio has been associated with clinical outcomes in several groups of cardiac patients, including patients with coronary artery disease, cardiac failure, and cardiac transplant recipients. We hypothesised that pre- and/or post-operative haematological cell counts are associated with clinical outcomes in children undergoing cardiac surgery for CHD. We performed a post hoc analysis of data collected as part of a prospective observational cohort study (n = 83, data available n = 47) of children evaluated for glucocorticoid receptor levels after cardiac surgery (July 2015-January 2016). The association of neutrophil-lymphocyte ratio with low cardiac output syndrome, time to inotrope free, and vasoactive-inotropic score was examined using proportional odds analysis, cox regression, and linear regression models, respectively. A majority (80%) of patients were infants (median/interquartile range 4.1/0.2-7.6 months) with conotruncal (36%) and left-sided obstructed lesions (28%). Two patients required mechanical circulatory support and three died. Higher pre-operative neutrophil-lymphocyte ratio was associated with higher cumulative odds of severe/moderate versus mild low cardiac output on post-operative day 1 (odds ratio 2.86; 95% confidence interval 1.18-6.93; p = 0.02). Pre-operative neutrophil-lymphocyte ratio was not significantly associated with time to inotrope free or vasoactive-inotrope score. Post-operative neutrophil-lymphocyte ratio was also not associated with outcomes. In children after congenital heart surgery, higher pre-operative neutrophil-lymphocyte ratio was associated with a higher chance of low cardiac output in the early post-operative period. Pre-operative neutrophil-lymphocyte ratio maybe a useful prognostic marker in children undergoing congenital heart surgery.

Validation and application of an HPLC-EC method for analysis of coenzyme Q10 in blood platelets.

Previous studies have indicated that analysis of coenzyme Q10 (CoQ10) in platelets may be clinically useful. The study objectives are to describe, validate and provide application of an HPLC-EC method for platelet CoQ10 analysis. This method analyzes oxidized (ubiquinone-10) and reduced (ubiquinol-10) forms of CoQ10 using two separate injections with the electrochemical analytical cell set at neutral and oxidizing potentials. Results showed that chromatograms were free of interfering peaks. Calibration curves were constructed over a concentration range 116-2317 nmol/L (r(2) = 0.99). The extraction recovery was >95%. The within-run precision CV% was < or =4.2%, and the day-to-day precision was < or =9.9%. Platelets were isolated by differential centrifugation, and frozen at -70 degrees C until analysis. The application of the method was used to compare accumulation of CoQ10 in platelets vs plasma in eight adult volunteers during a 28 day supplementation period (5 mg/kg/day of ubiquinol-10). Mean platelet total CoQ10 was 164 pmol/10(9) cells, and ubiquinol-10:total CoQ10 ratio was 0.56. During supplementation platelet CoQ10 levels were more consistent and predictable than plasma CoQ10 levels. The results indicate that this validated method for platelet ubiquinol-10 and ubiquinone-10 analysis is acceptable for use in the clinical laboratory, and that platelet CoQ10 may have important advantages over plasma during CoQ10 supplementation.

Systematic evaluation of muscle coenzyme Q10 content in children with mitochondrial respiratory chain enzyme deficiencies.

Coenzyme Q10 content, pathology evaluation, and electron transport chain (ETC) enzyme analysis were determined in muscle biopsy specimens of 82 children with suspected mitochondrial myopathy. Data were stratified into three groups: "probable" ETC defects, "possible" ETC defects, and disease controls. Muscle total, oxidized, and reduced coenzyme Q10 concentrations were significantly decreased in the probable defect group. Stepwise logistic regression indicated that only total coenzyme Q10 was significantly associated with probable ETC defect. Receiver operator characteristic (ROC) analysis suggested that total muscle coenzyme Q10 was the best predictor of an ETC complex abnormality. Determination of muscle coenzyme Q10 deficiency in children with suspected mitochondrial disease may facilitate diagnosis and encourage earlier supplementation of this agent.

Coenzyme Q10 (ubiquinol-10) supplementation improves oxidative imbalance in children with trisomy 21.

Endogenous coenzyme Q10 is an essential cofactor in the mitochondrial respiratory chain, a potent antioxidant, and a potential biomarker for systemic oxidative status. Evidence of oxidative stress was reported in individuals with trisomy 21. In this study, 14 children with trisomy 21 had significantly increased (P < 0.0001) plasma ubiquinone-10 (the oxidized component of coenzyme Q10) compared with 12 age- and sex-matched healthy children (historical controls). Also, the mean ratio of ubiquinol-10 (the biochemically reduced component):total coenzyme Q10 was significantly decreased (P < 0.0001). After 3 months of ubiquinol-10 supplementation (10 mg/kg/day) to 10 patients with trisomy 21, the mean ubiquinol-10:total coenzyme Q10 ratio increased significantly (P < 0.0001) above baseline values, and 80% of individual ratios were within normal range. No significant or unexpected adverse effects were reported by participants. To our knowledge, this is the first study to indicate that the pro-oxidant state in plasma of children with trisomy 21, as assessed by ubiquinol-10:total coenzyme Q10 ratio, may be normalized with ubiquinol-10 supplementation. Further studies are needed to determine whether correction of this oxidant imbalance improves clinical outcomes of children with trisomy 21.

Teaching pediatric laboratory medicine to pathology residents.

CONTEXT: Laboratory data are essential to the medical care of fetuses, infants, children, and adolescents. However, the performance and interpretation of laboratory tests on specimens from these patients, which may constitute a significant component of the workload in general hospitals and integrated health care systems as well as specialized perinatal or pediatric centers, present unique challenges to the clinical pathologist and the laboratory. Therefore, pathology residents should receive training in pediatric laboratory medicine. OBJECTIVE: Children's Health Improvement through Laboratory Diagnostics, a group of pathologists and laboratory scientists with interest and expertise in pediatric laboratory medicine, convened a task force to develop a list of curriculum topics, key resources, and training experiences in pediatric laboratory medicine for trainees in anatomic and clinical pathology or straight clinical pathology residency programs and in pediatric pathology fellowship programs. DATA SOURCES: Based on the experiences of 11 training programs, we have compiled a comprehensive list of pediatric topics in the areas of clinical chemistry, endocrinology, hematology, urinalysis, coagulation medicine, transfusion medicine, immunology, microbiology and virology, biochemical genetics, cytogenetics and molecular diagnostics, point of care testing, and laboratory management. This report also includes recommendations for training experiences and a list of key texts and other resources in pediatric laboratory medicine. CONCLUSIONS: Clinical pathologists should be trained to meet the laboratory medicine needs of pediatric patients and to assist the clinicians caring for these patients with the selection and interpretation of laboratory studies. This review helps program directors tailor their curricula to more effectively provide this training.

Clinical laboratory monitoring of coenzyme Q10 use in neurologic and muscular diseases.

Coenzyme Q10 (Q10) is available as an over-the-counter dietary supplement in the United States. While its use could be considered a form of alternative therapy, the medical profession has embraced the use of Q10 in specific disease states, including a series of neurologic and muscular diseases. Clinical laboratory monitoring is available for measurement of total Q10 in plasma and tissue and for measurement of redox status, ie, the ratio of reduced and oxidized forms of Q10. Many published studies have been anecdotal, in part owing to the rarity of some diseases involved. Unfortunately, many studies do not report Q10 levels, and, thus, the relationship of clinical response to Q10 concentration in plasma frequently is not discernible. Consistent laboratory monitoring of patients treated with this compound would help ease interpretation of the results of the treatment, especially because so many formulations of Q10 exist in the marketplace, each with its own bioavailability characteristics. Q10 has an enviable safety profile and, thus, is ideal to study as an adjunct to more conventional therapy. Defining patient subpopulations and characteristics that predict benefit from exogenous Q10 and defining therapeutic ranges for those particular applications are major challenges in this field.

Sensitized splenocytes result in deleterious cytokine cascade and hyperinflammatory response in rats with Pneumocystis pneumonia despite the presence of corticosteroids.

The immune response to the opportunistic pulmonary pathogen Pneumocystis can have beneficial and harmful effects on the host despite the presence of corticosteroids. We hypothesized that this deleterious hyperinflammatory response is associated with exaggerated cytokine production. The adoptive transfer of at least 10(7) immune splenocytes reduced the cyst count in rats with corticosteroid-induced pneumocystosis. About 18% of these rats developed clinical illness, an increased lung weight/body weight (LW/BW) ratio, and elevated levels of interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, tumor necrosis factor alpha, IL-5, IL-10, and gamma interferon in the lungs. This hyperinflammatory reaction was not observed in rats that remained clinically well or in control rats. Thus, in this model, corticosteroids have little effect on the cytokine cascade or other adverse effects of the host immune response to Pneumocystis.

New rat model of Pneumocystis pneumonia induced by anti-CD4(+) T-lymphocyte antibodies.

The CD4(+) T lymphocyte plays a central role in host defense against Pneumocystis pneumonia but has received only limited attention in rats. CD4(+) T-cell-depleting (OX-38) and nondepleting (W3/25) monoclonal antibodies, which recognize an identical or adjacent epitope, were administered for up to 14 weeks to Lewis rats that had been exposed to PNEUMOCYSTIS: While OX-38 produced a greater decrease in circulating CD4(+) cells than W3/25, both antibody treatments resulted in similar effects on the health of the rats and the levels of Pneumocystis pneumonia, which were milder than those found with corticosteroids. W3/25 also did not enhance the severity of Pneumocystis pneumonia achieved with corticosteroids alone. We conclude that CD4(+) cell function is more important than CD4(+) cell number in host defense against Pneumocystis in the rat and that this new model permits study of opportunistic infections in the rat without the confounding effects of corticosteroids.

Lymph node grading for staging of mycosis fungoides may benefit from examination of multiple excised lymph nodes.

BACKGROUND: Lymph node (LN) histopathologic class has been shown to be a significant determinant of survival in patients with mycosis fungoides. Often, histopathologic evaluation of just 1 node is used in staging patients with cutaneous T-cell lymphoma. OBJECTIVE: We examined whether sampling multiple nodes alters the staging and prognostic group placement of patients with mycosis fungoides as compared with sampling just 1 node. METHODS: Multiple LNs were obtained from a single, local region for histopathologic evaluation and grading in 8 patients with mycosis fungoides. RESULTS: Differences in histopathologic grading using multiple nodes were found in 5 of 8 patients. There was a potential upstaging of the assigned disease stage, compared with the stage that might have been assigned had just 1 node been sampled, in 3 patients. The differences in LN grading also potentially led to differences in prognostic group placement in 4 patients. CONCLUSION: Determining histopathologic grades from multiple LNs may allow a more accurate stage and prognosis to be assigned to patients.

Massive venlafaxine overdose resulted in a false positive Abbott AxSYM urine immunoassay for phencyclidine.

CASE REPORT: A 13-yr-old girl overdosed on 48 x 150 mg venlafaxine (Effexor XR). She was taking venlafaxine regularly for depression. Her only other medications included topical Benzamycin and pyridoxine 50 mg daily for acne. The Abbott AxSYM assay was positive only for phencyclidine, but GC/MS did not confirm the presence of phencyclidine. Toxilab identified only one substance, confirmed by GC/MS as venlafaxine. A serum sample obtained 3 h after her ingestion revealed a venlafaxine concentration of 24460 ng/mL and an O-desmethylvenlafaxine concentration of 3930 ng/mL, confirming the massive acute overdose (therapeutic range of venlafaxine and O-desmethylvenlafaxine together is 250-750 ng/mL). Urine spiked with 4.2 mg/mL ofvenlafaxine and 0.7 mg/mL of O-desmethylvenlafaxine was interpreted as positive with the Abbott AxSYM fluorescent polarized immunoassay for phencyclidine (readout of 28 ng/mL). CONCLUSION: Venlafaxine may cause a false positive Abbott AxSYM phencyclidine assay when present in very high concentrations. Physicians should be aware of this potential reaction when interpreting urine drug immunoassays.

Helicobacter pylori eradication in dyspeptic primary care patients: a randomized controlled trial of a pharmacy intervention.

OBJECTIVE: To determine the effectiveness of structured adherence counseling by pharmacists on the eradication of Helicobacter pylori when using a standard drug treatment regimen. DESIGN: Randomized controlled clinical trial. SETTING: Nonprofit group-practice health maintenance organization (HMO). PARTICIPANTS: HMO primary care providers referred 1,393 adult dyspeptic patients for carbon 14 urea breath testing (UBT). INTERVENTIONS: Those whose tests were positive for H pylori (23.3%) were provided a standard antibiotic regimen and randomly assigned to receive either usual-care counseling from a pharmacist or a longer adherence counseling session and a follow-up phone call from the pharmacist during drug treatment. All subjects were given the same 7-day course of omeprazole, bismuth subsalicylate, metronidazole, and tetracycline hydrochloride (OBMT). Dyspepsia symptoms were recorded at baseline and following therapy. OUTCOMES: The main outcome was eradication of H pylori as measured by UBT at 3-month follow-up. Secondary outcomes were patient satisfaction and dyspepsia symptoms at 3-month follow-up. RESULTS: Of the 333 participants randomly assigned to treatment, 90.7% completed the 3-month follow-up UBT and questionnaires. Overall eradication rate with the OBMT regimen was 80.5% with no significant difference in eradication rates between the 2 groups (P=0.98). Conclusions In this study, additional counseling by pharmacists did not affect self-reported adherence to the treatment regimen, eradication rates, or dyspepsia symptoms but did increase patient satisfaction.